DESCRIPTION: Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD), originally called the dsRNA unwinding/modifying activity, converts adenosines within dsRNA to inosines using a hydrolytic deamination mechanism. The enzyme has been detected in organisms throughout the animal kingdom, including humans. Recent evidence indicates that dsRAD is an RNA editing enzyme, but it remains possible that the enzyme has other biological functions as well. As an RNA editing enzyme dsRAD deaminates specific adenosines within mammalian glutamate receptor mRNAs and hepatitis delta virus antigenomic RNA resulting in codon changes that ultimately yield an altered protein. These editing events are a way to alter gene expression at specific times. The long term goal of the proposed research is to understand in as much detail as possible the intrinsic biochemical properties of dsRAD, and how these properties allow the enzyme to carry out its biological function. Recent evidence suggests the specificity needed for the patterns of editing observed in vivo is intrinsic to dsRAD and does not require the presence of other factors. Thus, careful studies of dsRAD's substrate specificity will provide clues as to how it promotes precise events in vivo. The proposed studies focus on how dsRAD recognizes its substrates, and once bound, how it chooses specific adenosines for modification. The in vitro studies will require purified dsRAD and dsRNA substrates. Previously developed protocols for purifying dsRAD will be used, and in addition, cloning and overexpression of dsRAD is planned. dsRAD's substrate specificity will be studied on synthetic substrates as well as the two recently identified biological substrates, glutamate receptor mRNA, and hepatitis delta virus antigenomic RNA. Experiments designed to identify additional dsRAD substrates are also proposed. The latter will employ techniques developed during the last grant period including a technique that allows selection of inosine containing RNAs using an anti-inosine antibody immunoaffinity column.